Dr.hani
rheumatic diseases
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rheumatic diseases are characterized by the
presence of one or more autoantibodies that may be
directe against components of the surface,
cytoplasm, nuclear envelope, or nucleus of the cell.
Immunofluorescence microscopy using human
cellular extracts, such as HEp-2 cells, allows for the
sensitive detection of serum antibodies that react
very specifically with various cellular proteins and
nucleic acids.
Systemic Rheumatic Diseases and Related
Disorders
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1.Systemic lupus erythematosus (SLE)
2. Discoid lupus erythematosus (DLE)
3. Lupus-like syndromes
4. Drug-induced lupus erythematosus
5. Sjogren's syndrome
6. Scleroderma/CREST syndrome (calcinosis cutis, Raynaud's phenomenon, esophageal
dysmotility, sclerodactyly, and telangiectasia)
7. Rheumatoid arthritis (RA)
8. Dermatomyositis and polymyositis
9. Overlap syndromes
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10. Systemic vasculitis
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a. Mixed Connective Tissue Disease (MCTD)
b. RAand SLE(Rupus)
c. SLEand scleroderma (Lupoderma)
d. Scleroderma and dermatomyositis (Sclerodermatomyositis)
e. Other
a. Takayasu's arteritis
b. Giant cell arteritis and polymyalgia rheumatica
c. Wegener's granulomatosis
d. Polyarteritis nodosa and Churg-Strauss syndrome
e. Leukocytoclastic vasculitis
f. Other
11. Poorly defined connective tissue disease syndromes
SLE
more than 110different types of
autoantibodies have been identified in SLE
 Widely used tests for screening of
intracellular autoantibodies are
immunofluorescence microscopy and the
immunoenzyme tests.The secondary
definitive tests for specific identification of
autoantibodies to nuclear antigens are
immunodiffusion, immunoprecipitation,
particle agglutination, enzyme-linked
immunosorbent assay(ELISA), and
immunoblolling methods.

SLE

SLE is characterized by a heterogeneous
and polyclonal antibody response, and the
usual case of SLE has an average of three
different circulating antibodies present
simultaneously, including antibody to native
DNA (dsDNA), chromatin. Sm antigen, U I
nRNP, SS-A/RO, SS-B/La, and several
other nonhistone protein or nonhistone
protein-RNA complexes .
SLE

specific for SLE:
 Anti-native-DNA (up to 90% of patients)
 anti-Sm
 anti-ribosomal-RNP
 and antibody to proliferating cell nuclear
antigen (anti-PCNA)
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Polyclonality of antibodies is seen in SLE
and scleroderma, and is rarely seen in the
other systemic rheumatic diseases.
Autoantibodies in SLE
Antibodies to cell nucleus component
ANA, anti-dsDNA, antibodies to extracellular nuclear
antigen (ENA, anti-Sm, anti-RNP, anti-Jo1)
 Antibodies to cytoplasmic antigens
anti-SSA, ANCA
 Cell-specific autoantibodies
lymphocytotoxic antibodies, anti-neurone antibodies,
anti-erythrocyte antibodies, anti-platelet antibodies
 Antibodies to serum components
antiphospholipid antibody anticoagulants
antiglobulin, rheumatoid factor
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Anti-nuclear antibodies(ANA)
Antinuclear antibodies (ANAs, also known as antinuclear factor or ANF) are
autoantibodies that bind to contents of the cell nucleus.
 There are many subtypes of ANAs such as:
 anti-Ro antibodies
 anti-La antibodies
 anti-Sm antibodies
 anti-nRNP antibodies
 anti-Scl-70 antibodies
 anti-dsDNA antibodies
 anti-histone antibodies
 Antibodies to nuclear pore complex
 anti-centromere antibodies
 anti-sp100 antibodies
 Each of these antibody subtypes binds to different proteins or protein complexes
within the nucleus. They are found in many disorders including autoimmunity, cancer
and infection, with different prevalences of antibodies depending on the condition.
 This allows the use of ANAs in the diagnosis of some autoimmune disorders,
including systemic lupus erythematosus, Sjögren's syndrome, scleroderma mixed
connective tissue disease polymyositis, dermatomyositis, autoimmune hepatitis and
drug induced lupus.

Serologic hallmarks of patients with systemic
autoimmune disease:
• SLE – sensitivity, 99 percent
• Scleroderma – 97 percent
• Mixed connective tissue disease – 93 percent
• Polymyositis/dermatomyositis – 61 percent
• Rheumatoid arthritis – 52 percent
• Rheumatoid vasculitis – 33 percent
• Sjögren's syndrome – 90 percent
• Drug-induced lupus –100 percent
• Discoid lupus – 15 percent
• Pauciarticular juvenile chronic arthritis – 71 percent
In Chronic infectious diseases (Mononucleosis, Subacute
bacterial endocarditis Tuberculosis ) and Other disorders (Some
lymphoproliferative diseases) and up to 50 percent of patients
taking certain drugs and can also be found in otherwise normal
individuals.
Anti-nuclear antibodies(ANA)
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
The common tests used for detecting and quantifying ANAs
are indirect immunofluorescence and enzyme-linked
immunosorbent assay (ELISA). In immunofluorescence, the
level of autoantibodies is reported as a titre
There are many nuclear staining patterns seen on HEp-2
cells(Human epidermoid cancer cells ):
 Homogeneous
 speckled
 Nucleolar
 nuclear membranous
 Centromeric
 nuclear dot
 pleomorphic.
ANA Screen 8 ELISA (IBL)
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dsDNA ………………………………plasmid……………………………………………SLE
RNP (proteins A, C, 68kDa) ……..human, recombinant ……….MCTD, SLE, RA, PSS
Sm (proteins B, B', D) ……………..bovine thymus …………………………………..SLE
SS-A/Ro (60kDa-proteAin)………..bovine thymus ……………………………..SS, SLE
SS-B/La human,…………………….recombinant ……………………………….SS, SLE
Scl-70 (DNA-topoisomerase I)……human, recombinant …………………………..PSS
CENP-B human,…………………….recombinant …………………………PSS (CREST)
Jo-1 (Histidyl-tRNA-synthetase) ….human, recombinant…………………………… PM
disadvantages of the ELISA
compared to IFA
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include a lack of sensitivity to unknown nuclear and
cytoplasmic antigens
the lack of an ANA pattern that has historically been reported
with the IFA
the diagnostic utility of an ANA of unknown specificity is not
clear at this time since many healthy people are ANA positive
but negative for diagnostically important autoantibodies
once a specific reactivity is identified in the serum, the ANA
pattern is no longer as important for diagnosis. Thus, the
ANA ELISA clearly has a useful place in a clinical laboratory.
Nucleolar proteins
Nucleolar
Ro
La
dsDNA
Smith
Rim
RNP
Jo-1
Speckled
Scl-70
Ro
Homogenous
Nucleosomes
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homogeneous pattern
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is seen when the condensed chromosomes and interphase chromatin stain.
 This pattern is associated with anti-dsDNA antibodies, antibodies to nucleosomal
components, and anti-histone antibodies.

There are two speckled patterns: fine and coarse.
 The fine speckled pattern has fine nuclear staining with unstained metaphase chromatin,
which is associated with anti-Ro and anti-La antibodies.
 The coarse staining pattern has coarse granular nuclear staining, caused by anti-U1-RNP
and anti-Sm antibodies.
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The nucleolar staining pattern is associated with many antibodies including anti-Scl70, anti-PM-Scl, anti-fibrillarin and anti-Th/To.
Nuclear membrane staining appears as a fluorescent ring around the cell nucleus
and are produced by anti-gp210 and anti-p62 antibodies.
The centromere pattern shows multiple nuclear dots in interphase and mitotic cells,
corresponding to the number of chromosomes in the cell. Nuclear dot patterns show
between 13–25 nuclear dots in interphase cells and are produced by anti-sp100
antibodies.
Pleomorphic pattern is caused by antibodies to the proliferating cell nuclear antigen.
Indirect immunofluorescence has been shown to be slightly superior compared to
ELISA in detection of ANA from HEp-2 cells
Antigen
Molecular Structure
Autoantibody
Frequency (%)
Native DNA
Double-strand DNA
40-90
Denatured DNA
Single-strand DNA
70
Histones
Hl,H2A.H2B,H3,H4
50-70
Chromatin(nucleosome)
DNA·histones complex
50-90
Sm
Proteins 29 (B'), 28 (B). 16 (D).and 13(E) 15-30
kDa, complexed with UI ,U2. and U4·U6
snRNAs; spliceosome component
Nuclear RNP (Ul nRNP)
Proteins 70, 33(A), and 22 (C) kDa.
complexed with Ul snRNA; spliceosome
component
30-40
SS-A/Ro
Proteins 60 and 52 kDa,
Complexed with Yl-Y5 RNAs
24-60
SS-B/La
Phosphoproteins 48 kDa,
complexed with YI nascenl RNA
Pol transcripts
9-35
Antigen
Molecular Structure
Autoantibody
Frequency (%)
Ku
Proteins 86 and 66 kDa.
DNA-binding proteins
1-19
hnRNP protein Al
Nuclear protein 34 kDa
31-37
PCNA
Protein 36 kDa; auxiliary protein
of DNA polymerase
3
Ribosomal RNP
Phosphoproteins 38. 16, and
IS kDa associated with ribosomes
10-20
Hsp·90
Heat-shock protein 90 kDa
5-50
Golgi complex
Golgins, giantin
Unknown
HMG-17
DNA-associated proteins, 9 to
17 kDa
34-70
B2-glycoprotein I
Anionic phospholipids. cardiolipin
25
Antibodies to Native DNA or
Double-Stranded DNA
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specific for SLE
40-90% in SLE
in 75-90% of active untreated SLE patients
Transient increases in anti-DNA antibodies were
recently described in RA patients treated with antiTNF therapy
reactive antibodies to DNA in the other diseases are
anti-single-stranded-DNA antibodies.
antibody to DNA is followed by the appearance of
circulating DNA antigen, Such DNA-anti-DNA
immune complexes, mostly containing complementactivating IgG3, have a special tropism for basement
membranes and are readily deposited in the kidney
glomeruli.
Hypothetical mechanism for the initiation of lupus
nephritis by basement membrane (BM)–binding antidouble-stranded DNA (dsDNA) antibody.
The stage I to II transition is likely to be reversible.62 The stage II to III
transition associated with the progressive accumulation of antibody and
chromatin into immune complexes will eventually reach a threshold for
which the immune complex deposition is no longer reversible. This
stage would yield chronic inflammation and lupus nephritis
Confocal micrographs of kidney cryosections
Co-localization of IgG with heparan sulfate proteoglycan (HSPG) is clearly
identified as turquoise
Antibodies to Native DNA or
Double-Stranded DNA
Earlier methods for detection of DNA
antibodies were the insensitive
precipitation method, complement
fixation, and passive hemagglutination.
 Current methods used are
radioimmunoassay (RIA). Indirect
immunofluorescence (IFA) on Crithidia
luci/iae, and enzymelinked
immunosorbent assay
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Negative staining of both kinetoplast and the nucleus.
Positive dsDNA :Staining of kinetoplast lying near the base of flagellum.
Ethidium bromide (red) added to show the nuclei of crithidia.
The problem with ELISAs is that they
detect both low and high avidity dsDNA
antibody (some ELISAs will also detect
single stranded DNA).
 High avidity anti-dsDNA antibodies are
specific for SLE associated nephritis
which is successfully detected by
crithidia and FARR assay.

Antibodies to Sm and Nuclear
Ribonucleoprotein(nRNP)

The spliceosomes consist
primarily of RNA-protein
complexes called small
nuclear ribonucleoproteins
(snRNPs). The snRNPs
are composed of small
nuclear RNAs (snRNAs) U1, U2, U4, U5 and U6 - as
well as a group of seven
proteins known as Sm
ribonucleoproteins that
collectively make up the
extremely stable Sm core of
the snRNP.
Antibodies to Sm and Nuclear
Ribonucleoprotein(nRNP)
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Highly specific
Sm and nRNP antigens were clearly associated,
because the nRNP could not be biochemically
isolated from Sm antigen
Sm and nRNP antigens were subcellular particles
comprised of small nuclear RNAs complexed with
proteins (at leastseven proteins varying in molecular
mass from 12 to 68 kDa).
found at a frequency of 30-40% in active SLE
No characteristic clinical feature,Patients who have
antibodies to only nRNP have a low frequency of
antibodies to DNA and a low frequency of clinically
apparent renal disease
Antibodies to Sm and Nuclear
Ribonucleoprotein(nRNP)
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Anti-Sm and anti-nRNP antibodies may be deteaed
by immunodiffusion, passive hemagglutination,
or counterimmunoelectrophoresis.(do not
accurately distinguish between antibodies
against different small nuclear-RNA-associated
polypeptides)
Immunoblotting has been found to be more
sensitive than conventional methods for the
detection of anti-Sm and anti-nRNP.
Currently, the most widely used laboratory tests
for the detection of anti-Sm and antinRNP
antibodies are immunodiffusion and
ELISA.(cannot define these pecific antibody
epitopes)
Ro/SSA ANTIGENS
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two cellular proteins with molecular weights of approximately 52
and 60 kD referred to as “Ro52” and “Ro60,”
Ro60 localized to the nucleus and nucleolus and with Ro52
localized to the cytoplasm.
Ro60 was shown to bind small, non-coding ribonucleic acids
(RNAs) termed “Y RNAs.” Although the function of Y RNAs is
unknown, one report suggests that Y RNAs may be a source of
micro (mi)RNA molecules, which have a role in the regulation of
messenger (m)RNA stability and translation
the protein can bind to both single- and double-stranded RNA.
The protein may function as a “RNA chaperone
Ro60 may also bind to other cellular and viral RNAs in the cell,
including Epstein-Barr virus (EBV) early RNA 1 (EBER1)
Ro52 :The protein localizes to the cytoplasm and functions as
an E3 ubiquitin ligase, an enzyme that adds ubiquitin molecules
to target proteins [4].
SSB/La ANTIGENS
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the SSB/La antigen was
highly concentrated in the
nucleolus of cells during the
late G1 and early S phase
and is thus cell cycle-related
Stimulate the activity of
polymerase alpha and beta
suggesting a further role in
elongation DNA
replication(associated with
RNA polymerase III
transcripts )
Role in DNA excision repair
Antibodies to SS-A/Ro and SS-B/La
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Patients with SLE can have antibodies to SS-A/Ro alone
or may have both anti- SS-A/Ro and anti-SS-B/La
anti-SS-A/Ro alone is strongly associated with HLA DR2
and with being young «22 years of age at onset).
both anti-SS-A/Ro and anti-SS-B/La in SLE is associated
with HLA-DR3 and is seen in older patients (>50 years
of age at disease onset)
patients with anti-SS-A/Ro alone had much more
serious renal disease
Anti SS-A/Ro autoantibodies have been closely
associated with the appearance of nephritis, vasculitis,
lymphadenopathy, photosensitivity and leukopenia in
SLE patients
Anti-SS-B/La antibodies, like anti-SS-A/Ro antibodies,
are antibodies noteworthy for their strong association
with Sjogren's syndrome.
Antibodies to SS-A/Ro and SS-B/La
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During pregnancy, anti-Ro antibodies can
cross the placenta and cause neonatal lupus
in babies
The 60kDa DNA/RNA binding protein and
52kDa T-cell regulatory protein are the best
characterised antigens of anti-Ro antibodies.
Collectively, these proteins are part of a
ribonucleoprotein (RNP) complex that
associate with the hyRNAs, hY1-hY5.
The La antigen is a 48kDa transcription
termination factor of RNA polymerase III,
which associates with the Ro-RNP complex.
Antibodies to SS-A/Ro

Elevated levels of SS-A/Ro antibodies
are related to several clinical
autoimmune disorders, including:
 (I) subacute cutaneous lupus erythematosus,
 (2) neonatal lupus erythematosus syndrome
with congenital heart block and cutaneous
lesions,
 (3) homozygous C2 and C4 deficiency with
SLE-Iike disease,
 (4) primary Sjogren's syndrome vasculitis,
rheumatoid factor positivity and severe
systemic symptoms,
 (5) ANA-negative SLE.
Pattern: SSB
Substrate: Hep2-cells
SPECKELED PATTERN
SSA
Fine fluorescence of nuclei sometimes surrounded by
„fluorescent dust“. This pattern is variable dependent on the
brand of Hep 2-cells
Anti-Ku
Ku-antigen system consists of a pair of proteins
called p70/p80
 DNA-binding proteins, interacting covalently
with the ends of native DNA
 found in 10% of 5LE sera and was not detected
in 100 scleroderma in immunoblot assays
 39% of SLE, 55% of mixed connective tissue
disease (MClD), and 40% of scleroderma patients
had low levels of antibody to Ku protein by elisa
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Anti-Ki
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first reported in Japan in 1981
In 10% of SLE patients
relationship was observed between anti-Ki and
the clinical features of arthritis, pericarditis,
and pulmonary hypertension in SLE patients
In ELlSA anti-Ki antibodies are observed in 21% of
SLE patients
while 7% were positive for anti-Ki by doubleimmunodiffusion tests.
Antibodies to P Ribosomal
Proteins
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Up to-20% of SLE sera show autoantibodies targeting
three ribosomal phosphoproteins of 15, 18, and 38
kDa
differences in the prevalence of anti-rRNP are found
among races.
occur in a higher frequency together with anti-sm and
anti-U 1nRNP antibodies in SLE
Association with major central nervous system disorders
in SLE, mainly psychotic symptoms,
No association was shown with cognitive impairment
or depression
Association with hemolytic anemia, leukopenia, alopecia,
malar rash, proteinuria, and neurologic complications
There was no correlation with disease activity.
Proliferating Cell Nuclear Antigen
PCNA complex has been characterized
as cell-cycle-related
 in 3% of patients with active SLE
 show no distinctive clinical associations
 A possible association with central
nervous system features, such as
transverse myelitis
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Antiphospholipid Antibodies
found in up to 60% of patients with SLE
 also in other disorders such as infectious
diseases
 The majority of anticardiolipin
antibodies cross-react with zwitterionic
(dipolar ion) phospholipids. Antibodies to
anionic phospholipids may be IgG or IgM,
whereas antibodies to zwitterionic
phospholipids are more frequently IgM.
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Antiphospholipid Antibodies
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Antibodies to phospholipids are identified in SLE
patients in three ways
 (1) serologically false-positive test for syphilis by a
positive Venereal Disease Research Laboratory
(VDRL) test, which is a flocculation assay using
carbon particles coated with cholesterol, lecithin
(phosphatidylcholine), and cardiolipin
 (2) the lupus anticoagulant assay, which is a
prolongation of the kaolin partial thromboplastin time
that is not corrected by normal plasma; and
 (3) cardiolipin immunoassay with the use of cardiolipin
or other negatively charged phospholipids as antigens
and B2-glycoprotein I as co-factor.

standard units (GPL and MPL) for anti phospholipid
antibody immunoassays:One GPL (MPL) unit is
equivalent to 1 mg/mL of an affinitypurified standard
IgG (lgM) sample
Antiphospholipid Antibodies
Newly developed assays for B2glycoprotein I autoantibodies seem to bring
specificity to the findings, since it was
described that the B2-glycoprotein is the
specific epitope for anti-phospholipid
autoantibodies
 Funhermore, IgAantibodies targeting B2glycoprotein I, or antibodies recognizing a
complex of B2-GP I/oxLDL (oxidized low
density lipoprotein) seem to be pathogenic
in atherosclerotic vascular disease
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Histone autoantibodies
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Histones are basic molecular proteins containing
high molar ratios of the positively charged amino
acids lysine and arginine.
The subunit of this histone-DNA complex is called a
nucleosome, which has two molecules of each of the
'core' histones (H2A, H2B, H3, and H4) and one
molecule of HI, along with DNA of about 200 base
pairs in length
Acetyltransferase is an enzyme that can acetyl ate
drugs such as hydralazine and procainamide,
playing an important role in detoxification and
excretion of the drug. Patients with low levels of
acetyltransferase were more prone to develop
ANA and clinical symptoms
Histone autoantibodies
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In drug-induced lupus, antibodies to single-stranded
DNA and histones are present. In SLE, on the other
hand, antibodies to double-stranded native DNA,
Sm, and UlnRNP antigens are often present
In the asymptomatic patients, the antihistone
antibody is predominantly IgM and displays broad
reactivity with all of the individual histones. Patients
with symptomatic disease develop a unique type of
IgG anti histone antibody, which, rather than reacting
with individual histones, shows specific reactivity with
the histone H2A-H2B dimer complex
Since anti histone antibodies are found in many
other conditions, more specific antibodies such as
antichromatin are replacing assays for antihistones
ANA Testing

Guideline #1 Test for autoantibodies
only when a consistent clinical suspicion
of rheumatic disease is present.
Not a good screening test for patients
with vague symptoms
 ANA can be positive in sick people (nonrheumatic) and healthy people

ANA Testing
Anti-Nuclear Antibodies, but they can
also be anti-cytoplasmic
 Immunofluorescence is commonly used
 In the past patients serum was placed
on to slides with rodent (or other animal)
cells and IF was performed to look for
antibodies binding to cellular
components
 What problems does this cause?

Human and rodent cells differ (slightly),
and so some people with obvious
rheumatic disease would be negative on
this test. “ANA-negative lupus”
 Now there are human tumor cell lines
that are used (HEp-2 are preferred)
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Another source of false negatives
includes how the tissues are fixed onto
the slides

Ethanol and methanol fixation may
remove Ro/SSA antigens from cells, so
the cells are fixed with acetone
How is the test done?
Patient serum is diluted and dropped
onto HEp-2 slides (cells fixed into
separate dots on the slide)
 Incubated, washed, secondary antibody
added
 Read by a tech using an IF scope (takes
specialized training and there is inherent
variability between individuals)

Results
Results typically include
positive/negative, titer and pattern of
staining
 Titers less than 1:40 should be
considered negative (20-30% of healthy
people)
 Titers of 1:40 to 1:160 should be
considered positive at low titer (further
workup is not recommended in the
absence of specific symptoms)

Results

Titers equal to or greater than 1:160
should be considered positive and
further workup should be done (only 5%
of healthy people). Prevalence of SLE
is 40-50 in 100,000 (but 5,000 will have
+ ANA)
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Each hospital can change these cutoffs
based on their patient population
What Follow-up Testing?
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Ideally this would depend on clinical
symptoms, but often:
dsDNA
Sm
nRNP
Ro/SSA
La/SSB
Scl-70
Jo-1
Patterns
The IF pattern is still reported, but does
not correlate well with what the
antibody’s specificity is.
 It was the most you could do “back in
the day”
 Now with ELISA testing for specific
antigens possible, the ANA pattern has a
low relevance

Patterns
Peripheral or rim = dsDNA
 Homogenous = dsDNA, histones
 Speckled = many antigens
 Nucleoli = associated with scleroderma
 Centromeric = CREST syndrome
 Cytoplasmic = myositis, mitochondrial


This homogenous
pattern of diffuse bright
green staining of nuclei
seen by
immunofluorescence
microscopy with a
Hep2 cell substrate is
called homogenous,
and is the most
common pattern with
autoimmune diseases
overall.
This rim (peripheral )
pattern of linear bright
green staining around
the peripheral of
nuclei seen by
immunofluorescence
microscopy with a
Hep2 cell substrate .
 dsDNA


Nucleolar pattern

Speckled pattern
SSA, SSB, Sm
Sp100_nuclear_antigen
Patterns

These little Crithidia
organisms have a
small kinetoplast
between the nucleus
and the flagella which
glows bright green
under
immunofluorescence
microscopy, and is
indicative of anti-native
DNA antibody that is
very specific for SLE.
To summarize…
You screen for ANAs using IF on slides
with HEp-2 cells
 If it’s positive you look for the specific
antigen that the antibody is reacting to
using ELISA (the antigen is stuck to the
well) or other methods
 We don’t screen for ANAs using ELISA
because it’s hard to get all the various
antigens (40+) onto the well walls

dsDNA
Guidelines suggest checking for antidsDNA antibodies only when the
symptoms are suspicious of SLE AND
the ANA is positive
The “ANA-negative lupus” patients are
REALLY rare now that we test with HEp2 cells rather than animal cells

Guidelines suggest that the only
antibodies that need to be quantified are
dsDNA (to predict a flare, and nephritis
risk)
 Active disease (q 6-12 weeks)
 Less active disease (q 6-12 months)
Report quantitative results on isolated U-RNP
antibodies (part of criteria for MCTD)
Thanks